Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex

Abstract

The vaccinia virus core contains a 195 kb double stranded DNA genome, a multi-subunit RNA polymerase, transcription initiation and termination factors and mRNA processing enzymes. Upon infection, vaccinia virus early gene transcription takes place in the virus core. Transcription initiates at early promoters and terminates in response to a termination motif, UUUUUNU, in the nascent mRNA. Early gene transcription termination requires the vaccinia virus termination factor, VTF, a single stranded DNA-dependent ATPase, and NPH I, the Rap94 subunit of the virion RNA polymerase, as well as the presence of the UUUUUNU motif in the nascent RNA. The position of UUUUUNU in the ternary complex suggests that it serves as a site of interaction with one or more components of the transcription termination complex. In order to identify the factor(s) that interact with UUUUUNU a series of direct UV photo crosslinking and ribonuclease A protection studies were undertaken. Through these analyses both VTF and Rap94 were shown to interact with UUUUUNU in the isolated ternary complex. Evidence indicates that the interaction is not mutually exclusive. VTF was shown to bind to UUUUUNU through the N-terminal domain of the large D1 subunit. Furthermore, VTF protects from RNAse A digestion both the 5' region of the nascent transcript as well as a large central component containing UUUUUNU. The addition of an oligonucleotide containing the (5Br)U9 sequence both directly inhibits transcription termination, in vitro and inhibits UV photo crosslinking of VTF to the nascent RNA in the ternary complex. These results support a model in which the availability of the UUUUUNU motif outside of the transcribing RNA polymerase permits binding of both transcription termination factors, VTF and Rap94, to UUUUUNU. The assembly of this termination complex initiates the transcription termination sequence.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

“…8). Based on cross-linking studies, Rap94, CE/VTF, and three subunits of vaccinia RNA polymerase (J6, A24, and D7) are arranged in the ternary complex such that they interact with the emerging nascent RNA (13). NPH I is bound to the transcription complex through an interaction between the C-terminal region of NPH I and the N-terminal region of Rap94 (19).…”

“…The transcription termination assay is similar to the transcript release assay described above except all four NTPs are included in the second step. Under these conditions, termination requires the presence of a U 5 NU signal in the nascent RNA and the viral CE, also known as VTF, in addition to NPH I (1,11,13,19,30).…”

“…This termination signal consists of U 5 NU (where N is any nucleotide) found 25-50 bases upstream of the 3Ј end of early transcripts (12). Capping enzyme, which is also known as vaccinia termination factor (VTF), has been shown to interact with the termination signal in the nascent RNA (13). NPH I is a single-stranded (ss) DNA-dependent ATPase that couples ATP hydrolysis with release of nascent RNA from the transcription elongation complex (TEC) (11,14,15).…”

Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant

“…Early gene transcription termination is a sequence specific event triggered by the sequence U5NU found 20-50 nt upstream of early gene 3′ ends (before polyadenylation) (Yuen & Moss, 1987). During viral early gene transcription termination, the sequence U5NU is recognized by CE via the N-terminal TPase domain of D1 (Christen et al, 2008). Termination itself is catalyzed by a DNA-dependent nucleoside triphosphate phosphohydrolase (NPHI, gene D11), which interacts with the early form of the RNA polymerase through contacts with the early gene specific H4 subunit (Deng & Shuman, 1996; Deng & Shuman, 1998; Christen et al, 1998; Mohamed & Niles, 2000).…”

Role of Forward Translocation in Nucleoside Triphosphate Phosphohydrolase I (NPH I)-mediated Transcription Termination of Vaccinia Virus Early Genes

“…Rap94 is an integral component of the virion form of vaccinia RNAP and is also required for early gene transcription through its association with NPH I (17). Furthermore, Rap94 was demonstrated to cross-link to the U 5 NU signal in the presence of VTF indicating that Rap94 may also play a direct role in termination (20). NPH I is required for release of the terminated nascent transcripts (15,16) and belongs to the DEXH-box family of helicases (16).…”